Figure 1

(A) Structure of JNJ-54271074. (B) Activity of JNJ-54271074 in 1-hybrid reporter assays. HEK-293T cells were transfected with RORγt, RORα and RORβ, which were fused with GAL4 DNA binding domain. After incubation with compound overnight, luciferase signals were measured. JNJ-54271074 was tested at a starting concentration of 1 μM for RORγt, and 6 μM for RORα and RORβ in 3-fold serial dilutions in duplicate. (C) FRET co-activator peptide assay. RORγt LBD was incubated with biotinylated TRAP220(631-655) as well as fluorescent donor and acceptor in the presence of titrated JNJ-54271074 and signals were measured at 665 nm and 615 nm (D) 2-hybrid co-repressor reporter assay. HEK-293T cells were transfected with NCoR and RORγt that was fused with NFκB activation domain in the presence of titrated JNJ-54271074. NCoR binding with RORγt triggers NFκB –driven luciferase signal. Representative dose response curves (of more than 30 experiments for the 1-hybrid assay, 4 experiments for the FRET assay and one experiment for the 2-hybrid assay) were plotted by percent activity of DMSO control vs. different concentrations, and presented as mean ± SEM of triplicate assays.